小鼠鳞状细胞细胞的肿瘤免疫分析及治疗反应评价
Summary
在这里, 我们提出了一个可重复的方法, 以开发一个原位小鼠模型的头颈部鳞状细胞癌。肿瘤表现出与临床相关的组织病理学特征的疾病, 包括坏死, 分化不良, 结节转移, 和免疫浸润。荷瘤小鼠出现临床相关症状, 包括吞咽困难、颌骨移位和体重减轻。
Abstract
头颈部鳞状细胞癌 (HNSCC) 是一种使人衰弱的致命疾病, 复发率高, 治疗失败。为了制定更好的治疗策略, 了解有助于治疗耐药的肿瘤微环境因素很重要。了解疾病机制和改进治疗的一个主要障碍是缺乏类似于人类 Hnscc 的攻击性和转移性的小鼠细胞系。此外, 大多数小鼠模型采用皮下植入肿瘤, 缺乏重要的生理特征的头部和颈部区域, 包括高血管密度, 广泛的淋巴血管, 和常驻黏膜菌群。本研究的目的是开发和表征 HNSCC 的正学模型。我们使用两个基因不同的小鼠细胞系, 并在小鼠的口腔黏膜中建立肿瘤。我们优化了基于胶原蛋白的肿瘤消化方法, 以最佳方式从已建立的肿瘤中恢复单个细胞。这里提供的数据表明, 小鼠发展高度血管化的肿瘤, 转移到区域淋巴结。单细胞多参数质量细胞学分析显示存在不同的免疫群, 骨髓细胞代表大多数免疫细胞。本研究提出的模型在癌症生物学、肿瘤免疫学和临床前新型治疗方法的发展中具有应用价值。正位模型与人类疾病的临床特征相似, 将为增强翻译和改善患者结局提供一个工具。
Introduction
HNSCC 是全球第五大最常见的恶性肿瘤, 每年诊断出60多万患者 1例。尽管积极治疗涉及化疗和放疗 ( RT ) , 但没有人类状瘤病毒 ( HPV ) 感染的 HNSCC 患者的总体生存率 ( OS ) 在 5 年后 2 年后仍低于 50% 。这主要是由于高度复杂的肿瘤微环境, 因为肿瘤可以来自头部和颈部区域内的几个不同的解剖部位, 包括口腔黏膜、舌头、口腔底部、鼻腔、口腔、咽部,口咽和下咽。此外, 头部和颈部区域是高度血管化, 并包含近一半的所有淋巴结在体内3。大多数研究头颈部肿瘤生物学的研究都依赖于侧翼区域的肿瘤模型。这样的模型可以提供深入了解肿瘤固有的机制, 但缺乏本地头部和颈部微环境可以显著影响这些发现的平移潜力。一种用于诱发口腔肿瘤的方法是接触致癌物质 9, 10-二甲基-2-苯甲氨基甲酮 (DMBA)4。然而, 这种方法是一个漫长的过程, 诱导肿瘤在大鼠和仓鼠, 但不在小鼠, 由此产生的肿瘤不具备许多组织学特征的分化 scc5,6。引入致癌物 4-硝基喹啉-氧化物 (4-NGO), 一种水溶性喹诺酮衍生物, 在口服时导致小鼠口腔肿瘤, 但也经历了长时间的接触时间 (16周), 批次内和批次之间的摄入率有限老鼠 7,8,9。为了开发临床相关模型, 几个小组使用了涉及操纵驾驶员癌基因或肿瘤抑制基因的基因工程模型, 包括 TP53、TGFB、KRAS、HRAS 和 SMAD410.这些模型可以提供对肿瘤的洞察与已知的驱动基因, 但不重述复杂的异质性的人类 Hnscc。
在这项工作中, 我们证明了在小鼠的水管内接种鳞状细胞癌细胞的可行性。注射细胞在注射后1周内发展成攻击性肿瘤。与人类 Hnscc 相似, 肿瘤转移到区域淋巴结。我们描述了该疾病的组织学和临床特征, 并提供了对肿瘤免疫微环境的洞察。我们认为, 这种 HNSCC 的原位模型在癌症生物学、肿瘤免疫学和临床前研究中具有应用价值。免疫逃避、肿瘤进展、治疗阻力和转移的机制是临床意义的领域, 可以使用该模型来解决。
Protocol
所有动物程序都是根据科罗拉多大学丹佛分校经批准的机构动物护理和使用委员会 (议定书 # 00250) 进行的。 1. 肿瘤细胞培养 请注意:B4B8 和 LY2 细胞系用于产生原位 HNSCC 肿瘤: B4B8 肿瘤细胞来源于致癌物质转化的黏膜角质形成细胞 (来自 balb 小鼠)11。Ly2 肿瘤细胞来源于自发转化的 balb 角质形成细胞系 (pam 212)12<s…
Representative Results
对 LY2 和 B4B8 细胞增殖的体外评估表明, 这两个细胞系的翻倍时间相似 (分别为21小时和 23小时)。在体内, 两个细胞系在接种后1周内形成一个单一的、可见的和明显的质量 (图 1a)。在患有 LY2 肿瘤的小鼠中, 由于肿瘤负担, 下颌被移位 3周 (图 1a)。控制没有接受肿瘤细胞的老鼠没有像预期的那样发展肿瘤。与 B4B8 肿瘤相比, LY2 肿瘤?…
Discussion
对肿瘤微环境进行严格的分析和表征, 是了解肿瘤发展、进展和转移机制以及开发有效治疗方法的重要策略。头颈部癌症是一种复杂的疾病, 可源于头部和颈部区域内的多个解剖部位。了解疾病机制和改进治疗的一个主要障碍是缺乏类似于人类 Hnscc 的侵袭性和转移性的小鼠细胞系。此外, 许多小鼠模型采用皮下植入肿瘤, 缺乏重要的生理特征的头部和颈部区域, 包括高血管密度, 广泛的淋巴血管, 和?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
没有
Materials
| Collagenase III | Worthington Biochemical Corp. | LS004183 | |
| DNase I | Worthington Biochemical Corp. | LS006328 | |
| Fc Block (CD16/32) | BD Biosciences | 553141 | Clone 2.4G2 |
| Flow Cytometry Staining Buffer | eBioscience | 00-4222-26 | |
| HBSS | ThermoFisher Scientific | 14175079 | no calcium, no magnesium, no pheno red |
| Helois mass cytometer | Fluidigm | NA | |
| Matrigel membrane matrix | Corning | CB-40234B | |
| MRI Scanner | Bruker | NA | 7.4 Tesla |
| RBC lysis buffer | BioLegend | 420301 | |
| Trypsin inhibitor | Worthington Biochemical Corp. | LS002830 |
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Cite This Article
Oweida, A. J., Bhatia, S., Van Court, B., Darragh, L., Serkova, N., Karam, S. D. Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment. J. Vis. Exp. (146), e59195, doi:10.3791/59195 (2019).